The retrotransposon Ty1 has served as a key model system for understanding the replication and integration of retroelements. In addition to providing key insights into replication mechanisms through ongoing mechanistic studies, it has become clear that Ty1 is an outstanding model for understanding the intimate relationship between retroelements and their host genomes. Replication of Ty1 elements is mediated by host RNA polymerase II, the Ty1-encoded reverse transcriptase (RT) enzyme, and a cellular tRNA primer, all acting on Ty1 RNA. Using a synthetic biology approach, we will produce a saturating map of the RNA and DNA structures required for retrotransposition. RNA apparently plays an as yet unclear role in retrotransposition, because Ty1 retrotransposition requires a several RNA processing enzymes including the RNA lariat debranching enzyme Dbr1. Integration of Ty1 cDNA is mediated by integrase (IN). Integration is targeted to very specific regions of the host genome, namely integration windows of several hundred base pairs immediately upstream of RNA polymerase III-transcribed genes. We are exploring this process through traditional genetic and biochemical studies as well as by using three new tools we have developed, 1) Design and analysis of synthetic Ty1 elements, 2) a Transposon Insertion Profiling chip (TIP-chip) and 3) ultra-high copy (UHC) Ty1 strains that contain more than 10 times the load of Ty1 elements carried by wild- type strains. The latter is an especially interesting microbial model for the relationship between retroelements and the genomes of higher eukaryotes, such as the human genome. A combined genetic, molecular and genomic approach will be taken to further dissect the Ty1 life cycle and Ty1's intimate relationship with its host genome.GM036481-A1 Project Narrative: The goal of this project is to deepen our understanding of how "jumping genes" change positions in a yeast cell's DNA. This mechanism is similar to the mechanism used by the HIV-1 virus (which causes AIDS) when it "jumps" into human DNA. Additionally, we study how the jumping genes interact with the machinery that protects our DNA from damage, a process that can lead to cancer.